The production of hydrogen sulfide by Saccharomyces cerevisiae during wine fermentation has long been a problem for wine makers as it has a low odour threshold.
Hydrogen sulfide can be formed metabolically by yeast from either inorganic sulfur compounds, sulfate and sulfite, or organic sulfur compounds, cysteine and glutathione. Cell growth creates a metabolic requirement for organic sulfur compounds, including cysteine, methionine, S-adenosyl methionine and glutathione. When these organic compounds are absent, the cell must synthesise them from inorganic sulfur compounds accumulated from the must. Under certain conditions, hydrogen sulfide is liberated during the reduction of inorganic sulfur to become detectable by the winemaker. The concentration of hydrogen sulfide produced varies with the availability of sulfur compounds, yeast strain and fermentation conditions, and the nutritional status of the environment. However, some strains appear to form unregulated amounts of hydrogen sulfide and presumably represent metabolic defects, at least in the wine environment (Mendes-Ferreira et al. 2002).
In Saccharomyces cerevisiae, hydrogen sulfide is the product of the Sulfate Reduction Sequence (SRS) pathway and acts as an intermediate in the biosynthesis of sulfur-containing amino acids. The ability of a strain to produce hydrogen sulfide is, at least, partly genetic, since hydrogen sulfide production by different wine strains varies under the same conditions (Henschke and Jiranek 1991, Jiranek et al. 1995a, Jiranek et al. 1995b, Jiranek et al. 1996). Mendes-Ferreira et al. (2002) recently screened a large selection of commercial wine yeast, in addition to non-Saccharomyces yeasts, which, when tested under identical physiological conditions, all had the same growth characteristics but varied in sulfite reductase (the enzyme producing hydrogen sulfide) activity. After fermentation in grape musts, yeast strains could be classified as nonproducers of hydrogen sulfide, must composition-dependent producers and invariable producers (Mendes-Ferreira et al. 2002).
The first step of the SRS metabolic pathway involves the transport of sulfate from the medium into the yeast cell via sulfate permease (FIG. 1). Sulfate is then reduced to sulfide through a series of steps using the enzymes ATP-sulfurylase (using two ATP molecules) and sulfite reductase. Sulfite reductase is a heterotetramer, consisting of two α- and two β-subunits, which are encoded by MET10 and MET5 genes, respectively. The enzyme, a hemoflavoprotein, binds the cofactors flavin adenine dinucleotide, flavin mononucleotide and siroheme. The next step leads to the sequestering of the sulfide: O-acetylserine (from the amino acid serine) combines with sulfide to form cysteine, and O-acetylhomoserine (from the amino acid aspartate) to form homocysteine, which can then be converted to methionine.
The problem of hydrogen sulfide production during wine making can be dealt with through the use of copper (which results in the formation of copper sulfide) or aeration (resulting in oxidation of the sulfide). Nevertheless, elimination of the use of copper salts by wineries is a desirable food processing goal and the presence of oxidised sulfur compounds in young wine could be related to the reductive character in bottled wine. Recent studies have therefore turned to investigating genetic methods for reducing hydrogen sulfide production.
One particular study (Spiropoulos and Bisson 2000) has investigated the role of the bifunctional O-acetylserine/O-acetylhomoserine sulfhydrylase as means to modulate hydrogen sulfide production by industrial yeast. Overexpression of the MET17 gene, which encodes O-acetylserine/O-acetylhomoserine sulfhydrylase, in a strain of Saccharomyces cerevisiae resulted in greatly reduced hydrogen sulfide formation. However, this was not the case with another strain, indicating that O-acetylserine/O-acetylhomoserine sulfhydrylase activity is not directly related to hydrogen sulfide formation.
Linderholm and Bisson (2005) have also evaluated the role of the sequence and level of expression of genes immediately downstream of sulfite reductase encoded by MET17, MET6 and CYS4. The genes were overexpressed in laboratory and brewing strains, but there was no universal reduction in hydrogen sulfide production. These genes were also sequenced in 12 wine isolates of this yeast. The MET17 alleles were identical in sequence to each other and to the sequence of the standard laboratory strain, S288C. For one additional commercial strain, a disruption of one of the MET17 alleles was found, but the other allele was identical to the consensus sequence. All 12 strains showed the identical five neutral base pair changes in CYS4 sequence when compared to the sequence reported for S288C. One strain contained an additional base pair change that led to an amino acid change. Two neutral base pair changes were observed in the sequences of MET6 for one wine yeast strain and three other strains had changes in sequence that were not neutral and altered the amino acid sequence. Genes encoding different alleles were used to transform a corresponding null mutation of S288C and enzyme activity and hydrogen sulfide production evaluated. The CYS4UCD932 allele resulted in faster fermentation rates and reduced hydrogen sulfide production when compared with the same strain transformed with CYS4S288C. The MET6 alleles showed no effects on sulfide formation in a null background.
Overexpression of the two genes MET14 and SSU1 have been shown to increase the formation of sulfite (Donalies and Stahl 2002). It has therefore been postulated that the deletion of the MET14 adenosylphosphosulphate kinase gene or the MRX1 methionine sulfoxide reductase gene might be the most effective way to prevent wine yeast from producing hydrogen sulfide (Pretorius and Bauer 2002, Pretorius 2003, 2004).
Modification of industrial yeast strains, particularly brewing and wine yeast strains, to reduce hydrogen sulfide production is still a highly desirable goal and the subject of ongoing investigations.